SUMMARY OF WORK: The ATP-dependent ion pumps generate transmembrane cation gradients which are essential for electrical excitation, solute transport, cell size and shape regulation and cation homeostasis. The Na+/K+-ATPase in mammalian cell membranes utilizes the energy available from ATP hydrolysis to transport 3 Na+ out of and 2 K+ into the cell during each catalytic cycle. The unequal stoichiometry of Na+ efflux and K+ influx results in an outward-flowing positive current which can be measured with a sensitive electrical recording device and correlated with the energy-yielding, enzymatic reactions. Using the laser flash/artificial bilayer technique and quenched-flow mixing, we measured the kinetics of the E1P(Na3)to E2P(Na3) conformational transition which is coupled to reorientation of the Na+ transport sites from the cytoplasmic to the extracellular surface and is presumed to be electrogenic (charge- translocating). In two different Na+/K+-ATPase preparations (eel electric organ, pig kidney) the rate constant for the phosphoenzyme conformational transition evaluated from the quenched-flow studies was 3 times larger than the rate constant determined from the electrical measurements. This suggests that there is an additional step in the transport cycle following the conformational transition that controls the kinetics of charge translocation (Na+ deocclusion?). Binding of Na+ and K+ involves similar amino acid residues in the a subunit, and when Na+ is released at the extracellular surface K+ is able to bind, activating the hydrolysis of E2P (to E2.Pi). We used the known rate constants and kinetic modeling to compare the time courses of electrogenic Na+ release and E2.Pi formation during the first turnover and discovered that K+ binds to the enzyme before Na+ is released. To resolve this paradox, we proposed that 2 Na+ ions are released immediately following the transition, creating 2 negatively-charged sites which bind extracellular K+, activating dephosphorylation (E2.Pi formation). This exchange is electrically silent because charge is conserved when K+ replaces Na+. After K+ is bound, but before it is transported into the cell, the third and final Na+ ion is released to the extracellular surface in a reaction that produces an outwardly- directed positive current. This sequence differs from an earlier model deduced entirely from voltage perturbation measurements in which the first Na+ ion released is electrogenic and is followed by release of the second and third Na+ ions before any K+ is bound.